Effect of Vasoactive Intestinal Peptide on the Necrotic or Apoptotic Neuronal Death in Murine Cortical Cell Cultures

ABSTRACT

OBJECTIVE: The purpose of this study was to examine the effects of VIP on the apoptotic neuronal death induced by serum deprivation and the necrotic(excitotoxic) neuronal death by the exposure of NMDA in primary murine cortical cell cultures. MATERIALS AND METHODS: Near-pure neuronal cell cultures were prepared by plating fetal mice cortical cells onto polyethyleneimine-coated 24 well vessels. At 7 days in vitro(DIV), serum was removed from culture media for 24 hrs in near-pure neuronal cultures. Mixed cortical cell cultures containing both neurons and glia were prepared by plating fetal mice cortical cells onto an established monolayer of glia. At 12-14 DIV, excitotoxic neuronal death was induced by the addition of NMDA into the mixed cortical cultures. The neuronal cell death was assessed by either MTT or LDH assay after microscopic examination. RESULTS: Near-pure neuronal cell cultures deprived of serum undergo neuronal apoptosis marked by cell body shrinkage, chromatin condensation and DNA ladders. The apoptotic neuronal death following serum deprivation was significantly attenuated by the inclusion of a membrane-permeable cAMP analogue(8-bromo-cAMP; 100nM), an adenyl cyclase stimulator(forskolin; 10nM), a protein synthesis inhibitor(cycloheximide; 0.1ng/ml) or cell membrane depolarization(25mM KCl) during serum deprivation, but was not affected by the addition of an NMDA antagonist (MK-801; 10nM) or an antioxidant(trolox; 100nM). The inclusion of VIP(1, 3, 10nM) during deprivation also significantly prevented the neuronal death without dose-dependency. The neuroprotective effect of VIP(1nM) was not reversed by concomitant treatment with a VIP receptor antagonist([D-p-Cl-Phe6, Leu17]-VIP; 10, 30nM). Neuronglia co-cultures exposed to 300nM NMDA for 5 min produced neuronal death marked by cell body swelling. The neuronal death induced by the exposure of NMDA was markedly attenuated by MK-801 but not affected by 8-bromo-cAMP, forskolin, cycloheximide, high potassium or VIP. CONCLUSION: These results provide an evidence that VIP prevent apoptotic neuronal death induced by serum deprivation and suggest that VIP may have therapeutic potential for diseases in central nervous system linked to apoptotic neuronal death.